Entamoeba gingivalis has been known for over 100 years, but because research has been limited, the role of this oral protozoan is still not yet delineated. Major reasons for the neglect of E.gingivalis are that it has been difficult to cultivate on artificial media and that it has never been grown as an axenic culture. We will start out by maintaining ATCC stock cultures of E.gingivalis with their associated undefined microflora on Diamond's TYGM-9 medium. E.gingivalis with their associated microflora will be transferred from the TYGM-9 medium into Modified Shaffer-Frye (MS-F) medium in order to initiate an amebae-bacteria monoxenic culture. Clostridium symbiosium and each isolate from the E.gingivalis stock culture will be added to separate MS-F cultures in order to determine which bacteria best supports the growth of E.gingivalis. An antibiotic mixture and differential centrifugation will be used to control and eventually eliminate the mixed flora of the inoculum. A 7-day MS-F culture will be introduced into TTY-SB monophasic medium and 2-3 million Crithidia per ml of medium from a 72-hour culture will be added in an effort to initiate an E.gingivalis-Crithidia monoxenic culture. An antibiotic solution and differential centrifugation will be used to eliminate the bacteria from the MS-F inoculum. The monoxenic culture of E.gingivalis grown in association with Crithidia species will be transferred from MS-F medium into Diamond's TYI-S-33 medium in an attempt to initiate an axenic culture. The predatory activity of the amebae and the temperature of incubation will lead to the elimination of the Crithidia species. Cultures will be considered axenic if they consist of E.gingivalis with no contaminants. If this method is not successful, we will attempt to establish an axenic culture with the aid of an Ecologen. E.gingivalis and the associated bacteria of the monoxenic culture will be grown in separate growth chambers, but they will be able to share cellular metabolites through a central diffusion reservoir. The monoxenic culture will be subjected to differential centrifugation in order to pellet the amebae. The pellet will then be used to inoculate the E.gingivalis growth chamber, and any associated bacteria will be eliminated by an antibiotic mixture. If the axenic cultivation of E.gingivalis can be established in the amebae growth chamber, then we will know that an axenic culture of E.gingivalis requires cellular metabolites produced by the bacteria. If the axenic cultivation of E.gingivalis can not be established, we will repeat the Ecologen procedure using Crithidia species instead of the bacteria from the MS-F culture.